1. Field
This disclosure is concerned generally with the testing of materials for the presence of pyrogens (e.g. bacterial endotoxins) and specifically with a pyrogenicity test for fat emulsion intended for intravenous administration.
2. Prior Art
Pharmaceutical solutions are routinely tested for the presence of pyrogens (fever producing substances) using either the U.S.P. rabbit test or, more recently, a coagulation test which involves use of a lysate of the amoebocyte blood cells of the horseshoe crab, Limulus sp. This latter test, commonly referred to as the Limulus lysate test, is based on the observation that the lysate will form a gel in the presence of very low concentrations (e.g. nanogram levels) of pyrogens such as bacterial endotoxins. The general Limulus lysate test procedure is described in detail in an article by E. Thye Yin et al, Biochem. Biosphys. Acta, Vol. 261, pp. 284-285 (1972). Various additives for improving the sensitivity of Limulus lysates are disclosed in a recent patent to R. E. Hopkins, U.S. Pat. No. 4,096,091.
Recently, pharmaceutically useful fat emulsions have become available to supplement intravenous solutions of various carbohydrates and amino acids. Although the carbohydrate and amino acid solutions can be tested for pyrogenicity using the U.S.P. rabbit test, or the Limulus lysate test, it has been found that when fat emulsions are tested by the U.S.P. rabbit test a false positive may occur as a result of a thermogenic response in the rabbits. See, for example, the study by G. F. Lambert et al, Am. J. Physiol. 164:490-496 (1951). See also articles by R. Grant, Amer. J. Physiol. 173:246-253 (1953) and S. W. Thompson et al, Amer. J. Clin. Nutrition 16:43-61 (1965).
We have found that the uncertainities (false positives) associated with using the U.S.P. rabbit test for fat emulsions can be avoided with a test procedure which, prior to our application, had not been associated with aqueous fat emulsions. Details of our test procedure, which appears to be more sensitive than the U.S.P. rabbit test on fat emulsions, are described below.